KR100577142B1 - A New Stress-Resistance Transcription Factor Gene, its Protein and A Transfected Plant by the Gene - Google Patents

A New Stress-Resistance Transcription Factor Gene, its Protein and A Transfected Plant by the Gene Download PDF

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KR100577142B1
KR100577142B1 KR1020030028792A KR20030028792A KR100577142B1 KR 100577142 B1 KR100577142 B1 KR 100577142B1 KR 1020030028792 A KR1020030028792 A KR 1020030028792A KR 20030028792 A KR20030028792 A KR 20030028792A KR 100577142 B1 KR100577142 B1 KR 100577142B1
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plant
capf1
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최도일
이소영
정영희
이상협
박정미
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한국생명공학연구원
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Abstract

본 발명은 식물병원체 및 환경스트레스에 의해 발현이 유도되는 EREBP/AP2-type 고추의 전사인자인 CaPF1에 관한 것이다.The present invention relates to CaPF1, which is a transcription factor of EREBP / AP2-type pepper, which is induced by phytopathogens and environmental stress.

본 발명은 CaPF1 전사인자를 암호화 하는 DNA 또는 상기 DNA를 내재하고 있어 식물체에 스트레스 저항성을 부여할 수 있는 식물체 형질전환용 발현벡터, 상기 발현 벡터에 의해 형질전환된 식물체 세포, 상기 상기 발현벡터에 의해 형질전환된 스트레스 저항성 식물체 개체 및 상기 스트레스 저항성 식물체 개체가 생산한 농산물을 제공한다. 또한 본 발명은, 상기 DNA를 식물체에 도입하여 식물체에 스트레스 저항성을 부여하는 방법을 제공한다.The present invention provides a DNA encoding the CaPF1 transcription factor or the expression vector for plant transformation capable of imparting stress resistance to the plant, the plant cell transformed with the expression vector, and the expression vector. Provided are transformed stress resistant plant subjects and produce produced by the stress resistant plant subjects. In another aspect, the present invention provides a method for introducing stress to the plant by introducing the DNA into the plant.

본 발명에 의하여 각종 생물적 또는 비생물적 스트레스에 대한 저항성을 가지는 유용 식물체를 간편하고 체계적으로 육종할 수 있게 된다.According to the present invention, it is possible to easily and systematically breed useful plants having resistance to various biological or abiotic stresses.

전사인자, 스트레스, 저항성, 식물, EREBP/AP2Transcription factor, stress, resistance, plant, EREBP / AP2

Description

스트레스 저항성 전사인자 유전자, 단백질 및 이에 의해 형질전환된 스트레스 저항성 식물체{A New Stress-Resistance Transcription Factor Gene, its Protein and A Transfected Plant by the Gene} A New Stress-Resistance Transcription Factor Gene, its Protein and A Transfected Plant by the Gene}             

도 1a는 병원성 세균을 접종한 고추 조직에서 질병 저항성에 따른 CaPF1의 발현을 보여주는 RNA gel 블럿 분석결과 사진.Figure 1a is a photograph of RNA gel blot analysis showing the expression of CaPF1 according to disease resistance in pepper tissues inoculated with pathogenic bacteria.

도 1b는 고추에서 CaPF1의 조직특이적 발현을 보여주는 RNA gel blot 사진.Figure 1b is a RNA gel blot photo showing tissue specific expression of CaPF1 in pepper.

도 2는 DraI, EcoRI, HindIII, 또는 XbaI으로 처리된 고추 총 DNA에 대해 CaPF1 cDNA를 탐침으로 한 게놈 DNA gel 블럿 분석결과 사진.Figure 2 is a photograph of genomic DNA gel blot analysis using CaPF1 cDNA probe for pepper total DNA treated with DraI, EcoRI, HindIII, or XbaI.

도 3은 생물적 스트레스에 의한 CaPF1의 발현을 보여주는 RNA gel 블럿 분석결과 사진.Figure 3 is a photograph of RNA gel blot analysis showing the expression of CaPF1 by biological stress.

도 4는 식물호르몬 처리 또는 환경 스트레스 처리에 의한 CaPF1의 발현을 보여주는 RNA gel 블럿 분석결과 사진.Figure 4 is a photograph of RNA gel blot analysis showing the expression of CaPF1 by plant hormone treatment or environmental stress treatment.

도 5는 CaPF1과 식물의 스트레스 저항성 관련 프로모터 서열인 GCC box 또는 CRT/DRE box와의 특이적 상호작용을 보여주는 사진.Figure 5 is a photograph showing the specific interaction of CaPF1 with GCC box or CRT / DRE box, a stress resistance related promoter sequence of plants.

도 6a, 6b는 애기장대에서 CaPF1의 과발현에 의한 방어-관련 유전자들의 항시발현을 보여주는 블럿결과 사진.6A and 6B are photographs of the results of blots showing the constant expression of defense-related genes by overexpression of CaPF1 in Arabidopsis;

도 7a, 7b, 7c는 CaPF1이 과발현된 담배에서 방어-관련 유전자들이 항시 발현되고, 식물 병원성 세균에 대한 저항성이 증가된 사실을 보여주는 블럿결과 사진, 개체사진 및 도표.7A, 7B, and 7C are photographs, individual photographs, and diagrams of blots showing the fact that defense-related genes are always expressed in CaPF1 overexpressed tobacco and resistance to plant pathogenic bacteria is increased.

도 8a, 8b는 CaPF1이 과발현된 애기장대에서 저온 스트레스 및 식물 병원성 세균에 대한 저항성이 증가된 현상을 보여주는 사진 및 도표.8a and 8b are photographs and diagrams showing the phenomenon of increased resistance to low temperature stress and plant pathogenic bacteria in Arabidopsis overexpressed CaPF1.

본 발명은 식물병원체 및 환경스트레스에 의해 발현이 유도되는 EREBP/AP2-type 고추의 전사인자인 CaPF1( C apsicum a nnuum Pathogen and Freezing tolerance related gene 1; 고추 병 및 저온 내성 관련 유전자 1)에 관한 것이다.The present invention relates to CaPF1 ( C apsicum a numum P athogen and F reezing tolerance related gene 1 ; pepper disease and cold resistance related gene 1), which is a transcription factor of EREBP / AP2-type pepper induced by phytopathogens and environmental stress. It is about.

식물은 생활사 동안 다양한 환경 스트레스를 받게 되고, 이는 올바른 생육에 좋지 않은 영향을 준다. 식물은 변화하는 환경에 대응하기 위해 적극적인 저항성 반응을 유도하는데, 그중 가장 흥미로운 것은 다양한 방어-관련 유전자들의 발현을 유도하는 것이다.Plants are subject to various environmental stresses during their lifetime, which adversely affects proper growth. Plants induce an aggressive resistance response to changing environments, the most interesting of which is the induction of expression of various defense-related genes.

특별히 전사인자들의 발현 변화는 스트레스 상황에 있는 식물에 많은 영향을 주게된다. 애기장대에는 DNA와 상호작용할 수 있는 Domain을 가지는 다양한 종류의 전사인자들이 있다. 이들 중에는 EREBP/AP2, bZIP, Myp, WRKY 그리고 Zinc finger 도메인을 갖는 단백질들이 포함되는데, 이들이 어떤 스트레스 상황에서 발 현이 유도되는가에 대한 연구가 활발히 진행 중에 있다(Rushton et al., 1998; Shinozaki and Yamaguchi-Shinozaki, 2000). 예를들어 토마토의 에칠렌 반응 인자(ETHYLENE-RESPONSE-FACTOR)인 Pti4가 과발현된 토마토는 식물의 곰팡이 병원체인 Erysiphe orontii와, 식물의 세균 병원체인 Pseudomonas syringae pv. tomato에 대한 저항성이 증가되었다. Pti4는 프로모터에 GCC box를 갖는 유전자들의 발현을 조절하는 기능을 가지고 있는 것으로 추정되고 있다(Gu et al., 2002; Wu et al., 2002). 또 다른 예로, 애기장대의 EREBP/AP2 type 전사인자인 CBP1/DREBP1과 DERBP1A를 과발현시킨 애기장대가 건조, 고농도의 염, 저온과 같은 환경 스트레스에 내성을 보인다는 연구결과가 보고되었다(Jaglo-Ottosen et al., 1998). 이들 두 전사인자는 저온 반응에 관여하는 프로모터 서열인 CRT/DRE box와 상호작용하고, target 유전자들의 발현을 조절하는 것으로 밝혀졌다(Kasuga et al., 1999). In particular, changes in the expression of transcription factors have a significant effect on plants under stress. Arabidopsis has a variety of transcription factors with domains that can interact with DNA. These include proteins with EREBP / AP2, bZIP, Myp, WRKY, and Zinc finger domains, and studies are underway in which stress situations they are expressed (Rushton et al., 1998; Shinozaki and Yamaguchi). Shinozaki, 2000). For example, tomato overexpressed with Pti4, the ETHYLENE-RESPONSE-FACTOR of tomato, is Erysiphe orontii , a fungal pathogen of plants, and Pseudomonas syringae pv. Resistance to tomato was increased. Pti4 is thought to have a function of regulating the expression of genes with GCC box in the promoter (Gu et al., 2002; Wu et al., 2002). As another example, studies have reported that Arabidopsis overexpressing the Arabidopsis EREBP / AP2 type transcription factors, CBP1 / DREBP1 and DERBP1A, are resistant to environmental stresses such as drying, high concentrations of salt, and low temperatures (Jaglo-Ottosen). et al., 1998). These two transcription factors have been shown to interact with the CRT / DRE box, a promoter sequence involved in low temperature reactions, and to regulate the expression of target genes (Kasuga et al., 1999).

담배의 에칠렌 반응 인자 중 하나인 Tsi1은 프로모터 서열인 GCC box와 CRT/DRE box 모두와 상호작용하며, Tsi1 유전자가 과발현된 담배가 병원성 세균 및 고농도의 염에 내성을 나타낸다고 보고되었다. 그러나 Tsi1 유전자가 과발현된 담배에서는 병에 대한 방어-관련 유전자들만 항시 발현되었을 뿐 내염성에 관련된 유전자의 항시 발현은 확인되지 않았다(Park et al., 2001).One of tobacco's ethylene response factors, Tsi1, interacts with both the promoter sequences GCC box and CRT / DRE box, and it has been reported that tobacco overexpressed with Tsi1 gene is resistant to pathogenic bacteria and high concentrations of salt. However, in cigarettes overexpressing the Tsi1 gene, only the defense-related genes for disease were always expressed, and the expression of the salt-related genes was not always confirmed (Park et al., 2001).

식물 호르몬과 같은 조절물질들은 서로 다른 신호전달체계에 관여한다. 살리실산, 에칠렌 그리고 자스몬산은 병원체 침입 후 병 저항성 신호전달 과정 중 이차 신호전달자로 활동하면서 많은 방어-관련 유전자들의 발현을 유도한다(Yang et al. 1997). 건조와 고농도의 염은 다량의 abscisic acid 생성을 유도하며, 외부에 서 처리한 abscisic acid는 건조와 저온 스트레스에 반응하는 여러 유전자들의 발현을 조절한다. 이러한 현상은 생물적 스트레스와 비생물적 스트레스에 관여하는 유전자들의 발현 유형이 서로 다르며, 이는 서로 다른 전사인자와 식물 호르몬의 서로 다른 조절의 결과이다. Regulators such as plant hormones are involved in different signaling systems. Salicylic acid, ethylene and jasmonic acid act as secondary signaling during pathogenic signaling following pathogen invasion, inducing the expression of many defense-related genes (Yang et al. 1997). Dry and high concentrations of salts induce the production of large amounts of abscisic acid, while externally treated abscisic acid regulates the expression of several genes in response to dry and cold stress. These phenomena differ in the types of expression of genes involved in biological and abiotic stresses, which are the result of different regulation of different transcription factors and plant hormones.

최근 연구결과, 생물적 스트레스와 비생물적 스트레스에 관여하는 신호전달 체계가 서로 공유되고 있다는 사실이 제시되고 있다. 첫 번째 증거는, 식물과 병원성 곰팡이 간의 비친화적 상호작용시 유도 발현되는 유전자 집단이 물리적인 상처에 의해 유도 발현되는 유전자 집단과 유사하다는 사실이다(Durrant et al., 2000). 두 번째 증거는, 유전자 칩 분석을 통해 곰팡이 병원균 접종 또는 건조 및 저온 스트레스 처리 후 발현이 유도되는 유전자의 무리가 매우 유사하다는 것이다(Schenk et al., 2000; Seki et al., 2001). 세 번째 증거는, 402개의 애기장대 전사인자로 이루어진 유전자 칩 분석 결과 서로 다른 스트레스 처리로 유도발현되는 유전자의 무리가 명확히 겹친다는 보고이다(Chen et al., 2002). 그러나 서로 다른 스트레스에 대응하는 식물의 저항성 기작이 상호 겹친다는 많은 연구에도 불구하고, 이에 관련된 분자생불학적 기작은 아직 밝혀지지 않았다. 따라서 식물의 생물적, 비생물적 스트레스 방어기작에 관련된 혹은 둘의 상호작용에 관련된 연구는 식물의 방어기작을 이해하는데 있어서 꼭 필요하다.Recent studies suggest that signaling systems involved in biological and abiotic stress are shared. The first evidence is that the gene pools that are induced and expressed during non-friendly interactions between plants and pathogenic fungi are similar to those that are induced and expressed by physical wounds (Durrant et al., 2000). The second evidence is that the gene chip analysis shows a very similar cluster of genes that are induced to express after fungal pathogen inoculation or dry and cold stress treatment (Schenk et al., 2000; Seki et al., 2001). A third piece of evidence is a report of gene chip analysis of 402 Arabidopsis transcription factors that clearly overlaps a herd of genes induced by different stress treatments (Chen et al., 2002). However, despite many studies that overlap the resistance mechanisms of plants that respond to different stresses, the molecular biologic mechanisms involved have not yet been identified. Thus, research relating to the biological and abiotic stress defense mechanisms of plants or their interactions is essential to understanding plant defense mechanisms.

본 발명은, 식물체에 스트레스 저항성을 부여하는 새로운 전사인자 유전자 및 그 단백질을 제공하는 것을 목적으로 한다.An object of the present invention is to provide a novel transcription factor gene and its protein that impart stress resistance to plants.

본 발명은, 식물체에 스트레스 저항성을 부여하는 새로운 전사인자 유전자로 형질전환된 스트레스 저항성 식물체를 제공하는 것을 또 다른 목적으로 한다.It is another object of the present invention to provide a stress resistant plant transformed with a new transcription factor gene that imparts stress resistance to the plant.

본 발명은, 식물체에 스트레스 저항성을 부여하는 새로운 전사인자 유전자로 식물체를 형질전환시켜 스트레스 저항성 식물체를 제조하는 방법을 제공하는 것을 또 다른 목적으로 한다.
Another object of the present invention is to provide a method for producing a stress resistant plant by transforming the plant with a new transcription factor gene that imparts stress resistance to the plant.

전술한 목적을 달성하기 위한 본 발명은, 고추 cDNA 라이브러리 (http://plant.pdrc.re.kr 참조)에서 추정적(putative) EREBP/AP2 type 전사인자 단백질을 암호화하는 신규의 유전자 CaPF1( C apsicum a nnuum Pathogen and Freezing tolerance related protein 1)을 분리하고 그 특성을 밝힌 사실에 기초한다.In order to achieve the above object, the present invention provides a novel gene CaPF1 ( C ) encoding a putative EREBP / AP2 type transcription factor protein in a pepper cDNA library (see http://plant.pdrc.re.kr). apsicum a nnuum P athogen and F reezing tolerance related protein 1 ) is based on the fact that it is isolated and characterized.

본 발명은, EREBP/AP2 도메인을 가지는 식물체 스트레스 저항성 유도 전사인자인 CaPF1을 암호화하는 DNA 또는 상기 DNA와 혼성화할 수 있는 DNA에 관한 것이다.The present invention relates to a DNA encoding CaPF1, which is a plant stress resistance inducing transcription factor having an EREBP / AP2 domain, or a DNA capable of hybridizing with the DNA.

이때 상기 DNA는 서열번호 1의 염기서열을 가지는 CaPF1 유전자 DNA일 수 있다. 또한 본 발명에서 상기 DNA는 서열번호 1의 염기서열과 80% 이상의 상동성을 가지는 DNA일 수 있다. In this case, the DNA may be CaPF1 gene DNA having a nucleotide sequence of SEQ ID NO: 1. In addition, the DNA in the present invention may be a DNA having a homology of 80% or more with the nucleotide sequence of SEQ ID NO: 1.

본 발명에서 상기 스트레스는 바이러스성, 세균성, 진균성 병원균과 같은 생물적(biotic) 스트레스나, 창상, 추위, 화학물질 등과 같은 비생물적(abiotic) 스 트레스를 모두 포함하는 개념이다.In the present invention, the stress is a concept that includes both biological stresses such as viral, bacterial, and fungal pathogens, or abiotic stresses such as wounds, colds, and chemicals.

또한 본 발명은 상기 각종의 DNA에 의해 암호화되는 식물체 스트레스 저항성 유도 전사인자 단백질에 관한 것이다. 예를들면 이들 전사인자 단백질은 서열번호 2의 아미노산 서열을 가지는 단백질이다.In addition, the present invention relates to a plant stress resistance induced transcription factor protein encoded by the various DNA. For example, these transcription factor proteins are proteins having the amino acid sequence of SEQ ID NO.

또한 본 발명은 상기 DNA를 내재하고 있어 식물체에 스트레스 저항성을 부여할 수 있는 식물체 형질전환용 발현벡터, 상기 발현벡터에 의해 형질전환된 식물체 세포 및 상기 발현벡터에 의해 형질전환된 스트레스 저항성 식물체 개체를 제공한다. 본 발명은 또한 상기 스트레스 저항성 식물체 개체가 생산한 농산물(예를 들면, 과일, 채소, 종자, 목재 등)에 관한 것이다.In addition, the present invention is a plant-transformation expression vector capable of imparting the stress resistance to the plant inherent the DNA, plant cells transformed by the expression vector and stress-resistant plant organisms transformed by the expression vector to provide. The invention also relates to agricultural products (eg, fruits, vegetables, seeds, wood, etc.) produced by the stress resistant plant individual.

또한 본 발명은, 상기 각종의 DNA를 식물체에 도입하여 식물체에 스트레스 저항성을 부여하는 방법과, 상기 각종의 병원균-반응성 전사인자 단백질의 발현을 변화시켜 식물체의 병저항성을 조절하는 방법을 제공한다.In another aspect, the present invention provides a method for imparting stress resistance to plants by introducing the various types of DNA into the plant, and a method for controlling plant disease resistance by changing the expression of the various pathogen-reactive transcription factor proteins.

이하 본 발명의 기술적 사상 및 본 발명의 기초가 되는 발견들에 대하여 상세히 설명한다.Hereinafter, the technical spirit of the present invention and the findings underlying the present invention will be described in detail.

본 발명에 의하면, CaPF1 유전자의 전사체는 숙주-비양립성(host-incompatible) 및 비숙주-비양립성(nonhost-incompatible) 병원균에 감염된 조직들에서뿐만 아니라, 에테폰(ethephon) 또는 자스몬산(jasmonic acid; JA)으로 처리된 잎에서도 유도되는 것으로 밝혀졌다.According to the present invention, the transcript of the CaPF1 gene is not only found in tissues infected with host-incompatible and nonhost-incompatible pathogens, but also by ethephon or jasmonic acid. (JA) was also induced in leaves treated with.

CaPF1의 과발현은 세균성 병원균에 대한 저항성을 나타내며, PR― 또는 환경 스트레스 방어관련―유전자들의 발현을 항시적으로 유도한다. 따라서 CaPF1로 형질전환된 식물체는 세균성 병원균 감염이나 저온 스트레스에 대하여 강한 저항성을 나타내었다. Overexpression of CaPF1 shows resistance to bacterial pathogens and induces expression of PR- or environmental stress defense-related genes at all times. Therefore, plants transformed with CaPF1 showed strong resistance to bacterial pathogen infection and low temperature stress.

EREBP/AP2 유형인 CaPF1 전사인자가 병원균의 감염, 에테폰 처리 또는 저온 처리에 의해 우선적으로 유도되므로, CaPF1 유전자가 생물적 혹은 비생물적 스트레스 방어 반응의 신호 전달 경로에 공히 관계된다는 사실을 추측케 한다. Since the CaPF1 transcription factor, an EREBP / AP2 type, is preferentially induced by pathogen infection, ethephon treatment, or low temperature treatment, it is speculated that the CaPF1 gene is involved in signaling pathways of biological or abiotic stress defense responses. do.

이하 CaPF1의 각종 특성과 관련하여 상세히 설명한다.Hereinafter, various properties of CaPF1 will be described in detail.

CaPF1은 고추에서 과민감 반응시 발현이 유도된다CaPF1 is Induced During Hypersensitivity in Red Pepper

과만감 반응 특이적 발현 유형을 보이는 유전자를 선별하기 위해 콩 불마름병 세균이 접종된 고추의 mRNA를 이용해 differential display 수행중 CaPF1이 선별되었다.CaPF1 was selected during differential display using mRNA of peppers inoculated with soybean blight bacteria to select genes with a specific overexpression specific expression type.

RNA 젤 블럿의 분석을 이용해 비숙주 비양립성(고추와 콩 불마름병균 상호작용), 또는 숙주 비양립성(고추와 고추 세균성 점무늬병균 상호작용) 상황에서 공통적으로 CaPF1의 발현이 유도되는 것으로 보아 CaPF1 유전자는 고추에서 세균에 의해 유도되는 과민감반응 유도부위에 특이적으로 유도발현 되는 사실을 알 수 있다.Analysis of the RNA gel blot suggests that CaPF1 genes are commonly induced in non-host incompatibility (pepper and soybean blight) or host incompatibilities (pepper and red pepper bacterial spot). It can be seen that is specifically induced expression of hypersensitivity reaction induced site induced by bacteria in red pepper.

담배 및 애기장대에서 CaPF1의 과발현은 방어관련 유전자의 활성화를 유도한다Overexpression of CaPF1 in Tobacco and Arabidopsis Induces Activation of Defense Related Genes

스트레스 방어작용에서 CaPF1의 기능을 분석하기 위해 CaPF1이 과발현되는 애기장대와 담배를 제조하였다. 두 서로 다른 종의 식물에서 공통적으로 방어관련 유전자가 활성화되었다. 생물적 스트레스 관련 방어유전자로 PR2, PDF1.2 그리고 GST1(애기장대, 담배) 유전자가 항시 발현되었고, 저온 트레스 관련 방어유전자로 COR47, COR6.6, 그리고, COR78(애기장대)이 항시 발현되었다. In order to analyze the function of CaPF1 in stress defense action, we have prepared Arabidopsis and Tobacco which overexpress CaPF1. Defense-related genes were activated in two different species of plants. The biotic stress related genes are expressed always been defensive genes PR2, PDF1.2 and GST1 (Arabidopsis, tobacco), COR47, COR6.6, and, COR78 (Arabidopsis thaliana), a low temperature stress defense-related genes were expressed at all times.

담배 및 애기장대에서 CaPF1의 과발현은 세균성 병원균에 대한 저항성을 유도한다Overexpression of CaPF1 in Tobacco and Arabidopsis Induces Resistance to Bacterial Pathogens

본 발명에서는, 담배 및 애기장대에서 CaPF1 유전자의 발현을 통해 방어기작에서 CaPF1의 기능을 밝혔다. In the present invention, the function of CaPF1 in defense mechanism through the expression of CaPF1 gene in tobacco and Arabidopsis.

CaPF1이 과발현된 애기장대의 경우 토마토의 세균병인 DC3000에 대한 저항성이 증가되었으며, CaPF1이 과발현된 담배의 경우 담배 세균성 불마름병균에 대한 저항성이 증가되었다. 이러한 현상은 CaPF1의 과발현에 의해 유도된 PR- 또는 방어-관련 유전자의 증가된 발현에 의한 것으로 추정되며, CaPF1 유전자가 세균에 대한 저항성 발생 경로에서의 커뮤니케이션에 중요한 역할을 하는 것으로 판단된다.Arabidopsis overexpressed CaPF1 increased resistance to DC3000, a bacterial disease of tomato, and increased resistance to tobacco bacterial blight bacteria in tobacco overexpressed CaPF1. This phenomenon is presumed to be due to increased expression of PR- or defense-related genes induced by overexpression of CaPF1 , and the CaPF1 gene is believed to play an important role in communication in the pathogenesis of resistance to bacteria.

애기장대에서 CaPF1의 과발현은 저온 스트레스에 대한 저항성을 유도한다Overexpression of CaPF1 in Arabidopsis Induces Resistance to Cold Stress

저온 내성 여부를 실험하기 위해 CaPF1이 형질전환된, 혹은 형질전환되지 않은 애기장대를 25℃에서 3주 키운 뒤 -5℃에서 24h 방치한 한 다음, 동사한 개체 수 측정했다. 형질전환되지 않은 애기장대는 90% 이상 동사했으며, CaPF1이 형질전환된 애기장대는 70%이상이 살아남았다.To test for low temperature resistance, CaPF1-transformed or untransformed Arabidopsis was grown at 25 ° C. for 3 weeks, left at −5 ° C. for 24 h, and then measured. More than 90% of the non-transformed Arabidopsis verbs were transfected, and more than 70% of the Arabidopsis transfected CaPF1 survivors.

이러한 사실로부터 CaPF1이 과발현된 애기장대의 경우 저온에 대한 내성이 증가됨을 알 수 있다.From this fact, it can be seen that the resistance to low temperature is increased in Arabidopsis overexpressed CaPF1.

이하 실시예에 의하여 본 발명을 보다 상세히 설명한다. 하기 실시예는 본 발명을 설명하기 위한 예시적인 것일 뿐 이에 의해 본 발명의 기술적 사상의 범위가 변경되거나 축소되는 것은 아니다.The present invention will be described in more detail with reference to the following examples. The following examples are merely illustrative for the purpose of describing the present invention, and thus the scope of the technical spirit of the present invention is not changed or reduced.

실시예 1 : CaPF1 유전자의 분리 분석Example 1 Isolation Analysis of CaPF1 Gene

식물의 스트레스 저항반응에서의 조절기작을 이해하기 위하여 고추 식물체로부터 CaPF1 유전자를 분리하고 그 특성을 분석하였다.To understand the regulatory mechanism of stress resistance in plants, CaPF1 gene was isolated and analyzed from pepper plants.

27±2℃, 16시간 명―8시간 암 조건에서 재배된 발아 8주령의 고추 식물체(Capsicum annunm cv. Bugang)를 모델시스템으로 이용하였다. Germinated 8-week-old pepper plants ( Capsicum annunm cv.Bugang) grown at 27 ± 2 ° C. and 16-hour dark-eight-hour dark conditions were used as a model system.

(1) 8주된 고추에 콩 불마름병균(Xanthomonas axonopodis pv. glycines 8ra ; Xag 8ra) 배양 혼탁액(1X108cfu/ml)과, 대조구로 1 mM MgCl2를 접종(syringe-infiltration)하고 소정의 시간마다 각 개체로부터 mRNA를 분리하여 세균병에 의해 유도되는 cDNA 라이브러리를 제작하였다. mRNA differential display에서 선별한 CaPF1 DNA조각을 탐침으로 이용해 full sequence를 갖는 클론을 분리했다. (1) 8 beans fire blight fungus to the main pepper (. Xanthomonas axonopodis pv glycines 8ra; Xag 8ra) culturing the suspension (1X108cfu / ml) and a 1 mM MgCl2 inoculation (syringe-infiltration) as the control, each at predetermined time MRNA was isolated from the individual to prepare a cDNA library induced by bacterial diseases. Clones with full sequence were isolated using CaPF1 DNA fragments selected from mRNA differential display as probes.

통상의 방법에 따라 상기 CaPF1 유전자 cDNA의 서열을 분석하였다(서열번호 1). 분석결과, CaPF1 유전자는 1.4kb의 핵산서열로 이루어져 있으며, 계산상 분자량이 41 KD인, 369개 아미노산 잔기로 이루어진 추정 아미노산 서열(서열 2)을 암호화하는 하나의 ORF(open reading frame)를 가지고 있는 것으로 밝혀졌다. The sequence of the CaPF1 gene cDNA was analyzed according to a conventional method (SEQ ID NO: 1). As a result, CaPF1 gene consists of 1.4kb of nucleic acid sequence and has one open reading frame (ORF) that encodes an estimated amino acid sequence (SEQ ID NO: 2) consisting of 369 amino acid residues with a calculated molecular weight of 41 KD. It turned out.

추정 아미노산 서열을 기초로 한 단백질 구조분석결과, CaPF1 단백질은 57개의 아미노산 서열로 이루어진 EREBP/AP2 DNA 결합 도메인과 핵으로 이동신호를 하는 짧은 염기성 잔기(basic residue) 및 산성 잔기들이 모여있는 도메인으로 구성되어 있었다. EREBP/AP2 도메인을 갖는 유전자 집단 중 아미노산 서열에서 가장 상동성을 보이는 것은 카드뮴에 의해 발현이 유도되는 것으로 알려진 애기장대의 AS30 유전자이다.As a result of protein structure analysis based on putative amino acid sequence, CaPF1 protein consists of EREBP / AP2 DNA binding domain consisting of 57 amino acid sequences and domains containing short basic residues and acidic residues that transfer signals to the nucleus. It was. The most homologous in the amino acid sequence among the gene population with EREBP / AP2 domain is the AS30 gene of Arabidopsis, which is known to be induced by cadmium.

(2) 전술한 (1)의 방법에 따라 소정 시간마다 CaPF1의 발현을 확인할 수 있도록 통상의 방법에 따라 RNA gel 블럿을 수행하였다(도 1A). 분석결과 도에서 볼 수 있는 바와 같이, 병원균이 접종된 경우, 시간이 경과함에 따라 CaPF1 유전자와 저항성 관련 유전자인 PR-4의 발현이 증폭되는 현상을 확인할 수 있었다. (2) RNA gel blots were performed according to a conventional method so as to confirm the expression of CaPF1 every predetermined time according to the method of (1 ) described above (Fig. 1A). As shown in the analysis results, when the pathogen was inoculated, it was confirmed that the phenomena of the expression of CaPF1 gene and PR-4 , a resistance-related gene, amplified with time.

(3) 여러 조직에서 CaPF1 전사체의 전사 특이성을 조사하였다. (3) The transcriptional specificity of CaPF1 transcript was examined in various tissues.

접종 18시간 경과 후, RNA 겔 블럿 분석을 위하여 종래 방법(Choi et al., 1996)에 따라, 감염된 고추 식물체의 뿌리, 줄기, 잎, 꽃 및 과실 조직 등 8개의 조직에서 총 RNA를 분리하였다. 각 샘플로부터의 총 RNA 20㎍을 포름알데하이드가 함유된 아가로스 겔 전기영동 방법(Sambrook et al., 2001)으로 분획하고, 나일론 멤브레인(Amersham, USA)으로 옮겼다. 분리된 RNA들을 블럿팅하였고, 블럿을 32P-표지된 전체 CaPF1 cDNA와 혼성화시켰다(도 1B). After 18 hours of inoculation, total RNA was isolated from eight tissues, including roots, stems, leaves, flowers and fruit tissues of infected pepper plants, according to conventional methods (Choi et al., 1996) for RNA gel blot analysis. 20 μg of total RNA from each sample was fractionated by agarose gel electrophoresis method with formaldehyde (Sambrook et al., 2001) and transferred to a nylon membrane (Amersham, USA). The isolated RNAs were blotted and the blots hybridized with 32 P-labeled total CaPF1 cDNA (FIG. 1B).

도에서 볼 수 있는 바와 같이, CaPF1 유전자에 대응하는 전사체는 꽃, 줄기, 발아한 종자에서 많이 발견하게 되었으나, 외부 스트레스 처리가 없는 잎 조직에서는 거의 발견되지 않았다. 보통 EREBP/AP2 유형의 전사인자들은 뿌리에서 발현량이 많은 것으로 알려져 있는데, CaPF1의 경우 뿌리에서의 발현 량이 매우 적었다.As can be seen in the figure, transcripts corresponding to the CaPF1 gene were found in flowers, stems, germinated seeds, but rarely found in leaf tissues without external stress treatment. Normally, EREBP / AP2 type transcription factors are known to have high expression in the roots, but CaPF1 has very little expression in the roots.

이상의 사실로부터 CaPF1 유전자는 조직 특이적으로 발현하는 것을 알 수 있다.From the above fact, it turns out that CaPF1 gene expresses tissue specific.

(3) 게놈 DNA gel blot 분석으로 고추 게놈 내에서 CaPF1의 카피수를 조사했다. (3) Genomic DNA gel blot analysis examined the copy number of CaPF1 in the pepper genome.

알려진 방법(Lee et al., 2002)에 따라, 고추 잎에서 게놈 DNA를 분리하였다. 20㎍ 게놈 DNA를 EcoRI, HindIII, XbaIDraI로 충분히 처리하였다. 처리된 게놈 DNA를 0.7% 아가로스 겔에서 전기영동하여 분리한 다음, 변성시키고 나일론 멤브레인(Amersham, USA) 상으로 블럿시켰다. 32P-dCTP로 표지된 CaPF1의 전체 cDNA 및 3'말단 부위의 유전자 특이적 서열 부분을 탐침자로 이용하여, 종래 방법(Church and Gilbert 1984)에 따라 서던 브롯팅을 수행하였다(도 2). 도에서 D, E, H 및 X는 각각 DraI, EcoRI, HindIII XbaI로 처리된 실험구를 의미한다.According to a known method (Lee et al., 2002), genomic DNA was isolated from pepper leaves. 20 μg genomic DNA was fully treated with EcoRI, HindIII, XbaI and DraI . Treated genomic DNA was isolated by electrophoresis on a 0.7% agarose gel, then denatured and blotted onto a nylon membrane (Amersham, USA). Southern blotting was performed according to the conventional method (Church and Gilbert 1984), using the whole cDNA and 3 'terminal region of the gene specific sequence of CaPF1 labeled with 32 P-dCTP as a probe (FIG. 2). D, E, H and X in the Figure means the experimental treatment treated with DraI, EcoRI, HindIII and XbaI , respectively.

도에서 볼 수 있듯이, 전체 cDNA를 이용한 분석에서는 5개의 밴드가, 3'말단 부위의 유전자 특이적 서열을 이용한 분석에서는 하나의 밴드가 관찰되었다. 위 분석결과로 미루어 보아 고추 게놈에는 EREBP/AP2 유형의 유전자가 여러 개 존재하고, CaPF1 유전자는 하나만 존재한다는 것을 알 수 있었다. As can be seen in the analysis, five bands were analyzed in the analysis using the whole cDNA, and one band was observed in the analysis using the gene specific sequence of the 3 'terminal region. As a result of the above analysis, it was found that there are several genes of EREBP / AP2 type and only one CaPF1 gene in pepper genome.

실시예 2 : 생물적 스트레스에 의한 CaPF1 유전자의 발현 분석Example 2 Analysis of Expression of CaPF1 Gene by Biological Stress

전기 실시예 1에서, 콩에 불마름병을 유발하면서 고추에는 비기주저항성을 유발하여 과민감 반응을 일으키는 콩의 불마름병균(X. axonopodis 8ra)을 접종했을 때, 고추에서 CaPF1 유전자의 발현이 유도됨을 확인하였다. 이러한 과민감반응 특이적 발현을 재확인하기 위해, (1X108cfu/ml)농도로 고추의 세균성 점무늬병균 Xanthomonas campestris pv. vesicatoria race 3(Xcv race3)을 로 민감성 고추 품종인 ECW (bs2/bs2) 및 저항성 품종인 ECW-20R (BS2/BS2)의 잎에 접종하여 과민감반응을 유도하였다. 상기 병원균은 avrBS2 유전자를 발현한다. 접종 후 다양한 시간대 별로 총 RNA를 추출하고 노던 블럿을 통해 CaPF1 유전자의 발현을 분석하였다(도 3). In Example 1, the CaPF1 gene expression was induced in peppers when inoculated with the soybean bacterium ( X. axonopodis 8ra), which induces hypersensitivity reactions to the peppers while inducing soybean disease. It was confirmed. To reconfirm this sense hypersensitivity reactions specific expression, (1X10 8 cfu / ml) concentration of bacterial strains of jeommunuibyeong pepper Xanthomonas campestris pv. vesicatoria race 3 ( Xcv race3) was inoculated into the leaves of sensitized pepper varieties ECW ( bs2 / bs2 ) and resistant varieties ECW-20R ( BS2 / BS2 ) to induce hypersensitivity. The pathogen expresses avrBS2 gene. Total RNA was extracted at various time points after inoculation and the expression of CaPF1 gene was analyzed by Northern blot (FIG. 3).

도에서도 볼 수 있듯이, 민감성 품종의 식물체(cv. ECW)에서는 36시간 경과 후 아무런 가시적 반응이 나타나지 않았지만, 저항성 고추 품종(cv. ECW-20R)에서는 24시간 이내에 CaPF1 유전자와 PR-4 유전자가 다량 전사되고, 과민감반응도 유도되었다.As can be seen, no visible response was observed after 36 hours in plants of sensitized varieties (cv. ECW), but large amounts of CaPF1 and PR-4 genes were detected within 24 hours in resistant pepper varieties (cv. ECW-20R). Transcription and hypersensitivity were induced.

실시예 3 : 다양한 비생물적 스트레스에 의한 CaPF1 유전자의 발현 분석Example 3 Analysis of CaPF1 Gene Expression by Various Abiotic Stresses

식물체가 비생물적(abiotic) 스트레스를 받을 때 CaPF1의 발현이 유도되는지를 확인하였다(도 4).It was confirmed whether the expression of CaPF1 is induced when the plant is subjected to abiotic stress (FIG. 4).

(1) 에칠렌, 살리실산, 자스몬산 등은 식물의 방어기작에관련된 신호전달체계에서 중요한 역할을 하는 호르몬으로 많은 연구가 되어있다. 위 호르몬들을 고추 잎에 분무한 후 CaPF1 유전자의 유도 발현 여부를 조사하였다. CaPF1 유전자는 에칠렌과 자스몬산에의 처리 30분만에 발현이 유도되었으나 SA에 의해서는 발현이 유도되지 않았다(도 4에서 A 참조).(1) Ethylene, salicylic acid, and jasmonic acid have been studied extensively as hormones that play an important role in signaling systems related to plant defense mechanisms. The hormones were sprayed on pepper leaves and examined for CaPF1 gene expression. Expression of CaPF1 gene was induced 30 minutes after treatment with ethylene and jasmonic acid, but not induced by SA (see A in FIG. 4).

(2) 고추에 대표적인 환경스트레스인 건조(0.4 M 만니톨 처리), 저온(4℃), 고농도의 염(0.4 M) 처리를 수행하여 CaPF1 유전자의 유도 발현 여부를 조사하였다. 처리한 모든 스트레스에 발현이 유도되었으나, 특별히 저온 스트레스 처리에 빠르고 강하게 유도 발현되었다(도 4에서 B 참조). (2) The pepper was dried (0.4 M mannitol), low temperature (4 ° C.), and high concentration of salt (0.4 M) to examine whether CaPF1 gene was induced. Expression was induced in all of the stresses treated, but was particularly fast and strongly induced expression in cold stress treatment (see B in FIG. 4).

실시예 4 : CaPF1 전사인자와 프로모터 서열 GCC box 또는 CRT/DRE box와의 특이적인 상호작용 여부 분석Example 4 Analysis of Specific Interaction of CaPF1 Transcription Factor with Promoter Sequence GCC Box or CRT / DRE Box

EREBP/AP2 유형의 전사인자들이 특이적으로 상호 작용하는 프로모터 서열은 이미 보고된 바 있다. 전사인자인 CaPF1 단백질도 방어관련 유전자의 프로모터 서열인 GCC box 또는 CRT/DRE box와 특이적인 상호작용을 하는지 여부를 확인하였다.Promoter sequences with specific interactions of EREBP / AP2 type transcription factors have already been reported. CaPF1 protein, a transcription factor, was also confirmed whether it specifically interacts with GCC box or CRT / DRE box, which are promoter sequences of defense-related genes.

이미 알려진 단백질 생산방법에 따라, CaPF1 유전자 말토스 결합 단백질(MBP) 유전자와 융합(MBP:CaPF1)시킨 뒤 대장균 내에서 발현시키고, 이를 순수 분리하여 gel retardation assay에 사용하였다. GCC box의 염기서열과 CRT/DRE box의 염기서열은 인공 합성하여 사용하였다(도 5). According to a known protein production method, the fusion with the CaPF1 gene maltose binding protein (MBP) gene (MBP: CaPF1) and then expressed in E. coli, it was purely isolated and used for gel retardation assay. The base sequence of the GCC box and the base sequence of the CRT / DRE box were used by artificial synthesis (Fig. 5).

실험 결과, 도 5에서도 볼 수 있는 바와 같이, CaPF1 단백질은 GCC box, CRT/DRE box와 공통적으로 상호작용 함을 알 수 있다. 종래 EREBP/AP2 유형의 단백질이 GCC box 또는 CRT/DRE box 둘 중 하나의 서열과 특이적인 상호작용을 하는 것에 비하여, 이러한 특성은 CaPF1 고유의 성질인 것으로 파악된다.As can be seen in Figure 5, it can be seen that CaPF1 protein interacts in common with the GCC box, CRT / DRE box. Compared to the conventional EREBP / AP2 type of protein that specifically interacts with the sequence of either the GCC box or the CRT / DRE box, these properties are found to be inherent to CaPF1.

실시예 5 : 애기장대와 담배에 CaPF1 과발현 효과 1 - 방어관련유전자와 저온반응 유전자들의 항시발현Example 5 Effect of CaPF1 Overexpression on Arabidopsis and Tobacco 1-Harmonal Expression of Defense Related Genes and Low Temperature Reaction Genes

CaPF1의 과발현과 방어관련유전자(PR gene) 및 저온반응 유전자(COR gene)와의 관련성을 분석하였다.Overexpression of CaPF1 and its association with defense gene (PR gene) and cold response gene (COR gene) were analyzed.

35S 프로모터를 갖는 pMBP1 벡터에 CaPF1 cDNA를 클로닝 하여 애기장대에 과발현시킨 후, 형질전환 제2 세대의 식물로부터 RNA를 분리하여 RNA gel blot 분석에 사용하였다. 이때 PR2, PDF1.2, 등 프로모터에 GCC box를 갖는 PR gene들과, COR47, COR6.6, COR78 등 프로모터에 CRT/DRE box를 갖는 COR gene들을 탐침으로 이용하였다. CaPF1 cDNA was cloned into pMBP1 vector with 35S promoter and overexpressed in Arabidopsis; RNA was isolated from the second generation of transformed plants and used for RNA gel blot analysis. At this time, PR2, PDF1.2, such as a promoter was used to probe the COR gene having a CRT / DRE box in such a promoter gene and PR, COR47, COR6.6, COR78 having a GCC box.

이러한 유전자 탐침들은 모두 외부 스트레스 처리 없이도 형질 전환된 애기장대에서 항시 발현되며, 고추와 같은 가지과 식물인 담배 형질전환체의 경우에도 외부스트레스 없이도 방어관련유전자(PR2, 3, 4, 5 gene)들이 항시 발현되며, 애기장대에서도 동일한 결과를 나타내었다(도 6A, 6B). 도에서 control은 빈 벡터만 형질전환시킨 negative control이고, line3, 8 및 22는 CaPF1을 형질전환시킨 제2 세대(따라서 CaPF1이 과발현된 애기장대)들 중 병과 저온 스트레스에 대한 내성이 상대적으로 우수한 개체를 의미한다.These gene probes are always expressed in Arabidopsis transformed without external stress treatment, and defense related genes (PR2, 3, 4, 5 genes) are always present even in the case of tobacco transformants, which are eggplants such as pepper, without external stress. It was expressed and showed the same result in Arabidopsis (FIGS. 6A and 6B). In the figure, the control is a negative control transforming only an empty vector, and lines 3, 8, and 22 are relatively superior to disease and low temperature stress among the second generations of CaPF1-transformed Arabidopsis overexpressed. Means.

위와 같은 결과는 전기 실시에 4에서 밝힌 특정 프로모터 서열(GCC box, CRT/DRE box)과 CaPF1단백질과의 상호작용이 생체 내(in vivo)에서도 동일하게 일어남을 확인하는 것이다.The above results confirm that the interaction between the specific promoter sequence (GCC box, CRT / DRE box) and CaPF1 protein in the previous embodiment 4 occurs in vivo .

실시예 6 : 애기장대와 담배에 CaPF1 과발현 효과 2 - 식물 병원성 세균 감염 및 저온 처리에 내성 증가 Example 6 CaPF1 Overexpression Effect on Arabidopsis and Tobacco 2-Increased Resistance to Plant Pathogenic Bacterial Infection and Cold Treatment

여러 가지 방어관련유전자가 항시 발현되는 CaPF1 과발현 형질전환체들(애기장대, 담배)이 보통의 식물과 비교해서 스트레스 내성이 증가 되었는지 여부를 분석하였다We analyzed whether CaPF1 overexpressing transformants (eggi pole, tobacco), which express various defense genes at all times, have increased stress tolerance compared to normal plants.

(1) 담배의 병원성 세균에 대한 저항성 증가 (1) increased tobacco resistance to pathogenic bacteria

담배 식물체에 세균성 점무늬병원균을 접종하고 그 결과를 분석하였다(도 7A, 7B, 7C). 도에서 c는 대조구(비형질전환체)를, 나머지 숫자는 형질전환된 클론의 종류를 의미한다.Tobacco plants were inoculated with bacterial spot pathogens and the results were analyzed (FIGS. 7A, 7B and 7C). In the figure c represents a control (nontransformer), the remaining number refers to the type of clone transformed.

도에서 볼 수 있듯이, 비형질전환체(대조구; c)에 비해 본 발명에 의한 CaPF1 유전자로 형질전환된 식물체의 경우 CaPF1 및 저항성 관련 유전자들의 전사가 크게 증가하였으며(도 7A), 육안관찰에 의하더라도 병증이 거의 나타나지 않았다(도 7B). 접종시간 경과에 따라서도 대조구에 비해 형질전환 식물체의 잎에서 병원균체 수가 매우 적게 나타났다(도 7C). 예를들면, 담배 식물체의 경우 비형질 전환체에 비해 조직내 살아있는 병원균 수가 100배 이상 감소되었다. As can be seen in the case of plants transformed with the CaPF1 gene according to the present invention, the transcription of CaPF1 and resistance-related genes was significantly increased compared to the non-transformant (control) c (FIG. 7A). Almost no symptoms were seen (FIG. 7B). Depending on the time of inoculation, the number of pathogens in the leaves of the transgenic plant was very low compared to the control (FIG. 7C). For example, tobacco plants have a 100-fold reduction in the number of viable pathogens in tissues compared to non-transformants.

이러한 사실은 CaPF1 전사인자의 과발현으로 담배 식물체가 세균병에 내성을 가지게 됨을 의미하는 것이다.This indicates that overexpression of CaPF1 transcription factors makes tobacco plants resistant to bacterial diseases.

(2) 애기장대의 저온 스트레스 및 병원성 세균에 대한 저항성 증가 (2) resistance to cryogenic stress and pathogenic bacteria in Arabidopsis;

CaPF1이 과발현된 애기장대를 24시간동안 -5℃에서 방치한뒤 살아남는 개체수를 측정한 결과 비형질전환체는 90%이상이 동사했고, 형질전환체는 70%이상의 개체가 살아남았다(도 8A). 도에서 Col-0는 대조구(비형질전환체)를 의미한다.After over-expressing the CaPF1 overexpressed Arabidopsis at -5 ° C for 24 hours, the surviving population was measured. As a result, more than 90% of the non-transformants and 70% of the transformants survived. . Col-0 in the Figure refers to the control (nontransformer).

또한 병원균 스트레스로서, 애기장대 식물체에 토마토의 병원성 세균인 DC3000을 접종하고 그 결과를 분석하였다(도 8B). In addition, as a pathogen stress, the Arabidopsis plants were inoculated with DC3000, a pathogenic bacterium of tomato, and the results were analyzed (FIG. 8B).

도에서 볼 수 있듯이, 비형질전환체(대조구; c)에 비해 본 발명에 의한 CaPF1 유전자로 형질전환된 식물체의 경우 육안관찰에 의하더라도 병증이 거의 나타나지 않았으며, 접종시간 경과에 따라서도 대조구에 비해 형질전환 식물체의 잎에서 병원균체 수가 매우 적게 나타났다. 예를들면, 형질전환된 애기장대의 경우 비형질전환체에 비해 조직내 살아있는 병원균 수가 10-100배 감소되었다. As can be seen in the case of the transformed plants with the CaPF1 gene according to the present invention compared to the non-transformant (control) c, the symptoms were almost no symptoms even by visual observation, even in the control group over time In comparison, the number of pathogens was very low in the leaves of transgenic plants. For example, the transformed Arabidopsis had a 10-100-fold reduction in the number of viable pathogens in tissues compared to nontransformants.

이러한 사실은 CaPF1 전사인자의 과발현으로 애기장대 식물체가 세균병에 내성을 가지게 됨을 의미하는 것이다.This suggests that overexpression of CaPF1 transcription factors makes Arabidopsis plants resistant to bacterial diseases.

본 발명에 의하여 각종 식물병원 세균 및 저온 스트레스에 대한 저항성을 모두 가질 수 있는 유용 식물체를 간편하고 체계적으로 육종할 수 있게 된다. According to the present invention, it is possible to simply and systematically breed useful plants that can have both resistance to various phytopathogenic bacteria and low temperature stress.                     

본 발명에서 이용되는 식물체에 스트레스 저항성을 부여하는 새로운 전사인자 유전자 및 그 단백질은 역사적으로 오랫동안 이용되어 왔던 작물인 고추에서 유래된 것이므로, 이를 이용하여 보다 안전한 형질전환 식물체 및 농산물을 얻을 수 있게 된다.Since the new transcription factor gene and its protein that impart stress resistance to the plant used in the present invention are derived from pepper, which has been used for a long time in history, it is possible to obtain safer transgenic plants and agricultural products.

또한 본 발명에 의해 식물체에서 외부 스트레스에 대한 저항 메카니즘이 규명되었기 때문에, 이후 관련 분야의 연구 및 기술개발에 기초를 제공할 수 있게 된다.
In addition, since the resistance mechanism against external stress in the plant has been identified by the present invention, it is possible to provide a basis for research and technology development in related fields.

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<110> Korea Research Institute of Bioscience and Biotechnology <120> A New Stress-Resistance Transcription Factor Gene, its Protein and A Transfected Plant by the Gene <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 1286 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of CaPF1 full length cDNA (1286 bp) <400> 1 atgtgtggtg gtgcaattat ctccgatttg gtacctccta gccggatttc ccgccggcta 60 accgccgagt tgctatgggg taactctgat ctgagcaaaa agaagaaaaa tccagggaat 120 tattactcaa agcctttgaa caggtctaag tttattgacc ttgatgagga atttgaagct 180 gactttcagg acttcaagga ctatgccgat gacgatgttg atgatgttaa gcccttcggt 240 tccaaatctg tgaaatctgg cgattcaagc tgcgatactg aaaaatcttc caagagaaag 300 aggaagaatc agtaccgggg gatcagacag cgtccttggg gtaagtgggc agctgaaatt 360 cgtgatccga ggaaagggat tcgagtttgg cttggaactt tcaattctgc ggaagaagca 420 gctagagctt atgatgttga ggcacgaagg atcagaggca agaaggctaa ggtgaacttt 480 cctgatggat ctccagcttc tgcttcaaga cgtgctgtta agccaaatcc tcaggaggca 540 cttcgcgagg aaatcttgaa cacagttcag ccgaacacaa cttatatcaa caacttggac 600 ggcggatctg atgattcgtt tggctttttc gaagagaaac cagcagcaaa gcagtatggc 660 tatgagaatg tttcttttac tgctggagat atgggactgg gttcaatttc cccttcaact 720 ggtacaacaa atgtttactt cagttctgat gaaggaagca acacctttga ctgctctgat 780 ttcggttggg gtgaaccatg tccgaggact ccagagatct catctgttct gtcagaagtt 840 ctagaatgta atggtactca atctgatgaa gatgctagac cagagaaaaa actgaagtcg 900 tgttccaacg cttccttgcc agatgaggat aacactgtgc acacgctatc tgaagagcta 960 tcggcttttg aatcccagat gaagttcttg cagatcccat atcttgaggg aaattgggat 1020 gcatcagttg atgcctttgt caacacaggc gcaattcagg atggcggaaa tgcgatggat 1080 ctctggcctt cgatgatgtt ccttctttaa tgggaggtgt ctataagcca acacgcacct 1140 tcccttatta agttttgtaa ataaagcttc atttgagtga agtttgcagt tatgttgtct 1200 ccaaacaaaa aagactatat atgtgttgta ttaaatttat ttcataaatt tacttgtttg 1260 atgtaaaaaa aaaaaaaaaa aaaaaa 1286 <210> 2 <211> 369 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of CaPF1 <400> 2 Met Cys Gly Gly Ala Ile Ile Ser Asp Leu Val Pro Pro Ser Arg Ile 1 5 10 15 Ser Arg Arg Leu Thr Ala Glu Leu Leu Trp Gly Asn Ser Asp Leu Ser 20 25 30 Lys Lys Lys Lys Asn Pro Gly Asn Tyr Tyr Ser Lys Pro Leu Asn Arg 35 40 45 Ser Lys Phe Ile Asp Leu Asp Glu Glu Phe Glu Ala Asp Phe Gln Asp 50 55 60 Phe Lys Asp Tyr Ala Asp Asp Asp Val Asp Asp Val Lys Pro Phe Gly 65 70 75 80 Ser Lys Ser Val Lys Ser Gly Asp Ser Ser Cys Asp Thr Glu Lys Ser 85 90 95 Ser Lys Arg Lys Arg Lys Asn Gln Tyr Arg Gly Ile Arg Gln Arg Pro 100 105 110 Trp Gly Lys Trp Ala Ala Glu Ile Arg Asp Pro Arg Lys Gly Ile Arg 115 120 125 Val Trp Leu Gly Thr Phe Asn Ser Ala Glu Glu Ala Ala Arg Ala Tyr 130 135 140 Asp Val Glu Ala Arg Arg Ile Arg Gly Lys Lys Ala Lys Val Asn Phe 145 150 155 160 Pro Asp Gly Ser Pro Ala Ser Ala Ser Arg Arg Ala Val Lys Pro Asn 165 170 175 Pro Gln Glu Ala Leu Arg Glu Glu Ile Leu Asn Thr Val Gln Pro Asn 180 185 190 Thr Thr Tyr Ile Asn Asn Leu Asp Gly Gly Ser Asp Asp Ser Phe Gly 195 200 205 Phe Phe Glu Glu Lys Pro Ala Ala Lys Gln Tyr Gly Tyr Glu Asn Val 210 215 220 Ser Phe Thr Ala Gly Asp Met Gly Leu Gly Ser Ile Ser Pro Ser Thr 225 230 235 240 Gly Thr Thr Asn Val Tyr Phe Ser Ser Asp Glu Gly Ser Asn Thr Phe 245 250 255 Asp Cys Ser Asp Phe Gly Trp Gly Glu Pro Cys Pro Arg Thr Pro Glu 260 265 270 Ile Ser Ser Val Leu Ser Glu Val Leu Glu Cys Asn Gly Thr Gln Ser 275 280 285 Asp Glu Asp Ala Arg Pro Glu Lys Lys Leu Lys Ser Cys Ser Asn Ala 290 295 300 Ser Leu Pro Asp Glu Asp Asn Thr Val His Thr Leu Ser Glu Glu Leu 305 310 315 320 Ser Ala Phe Glu Ser Gln Met Lys Phe Leu Gln Ile Pro Tyr Leu Glu 325 330 335 Gly Asn Trp Asp Ala Ser Val Asp Ala Phe Val Asn Thr Gly Ala Ile 340 345 350 Gln Asp Gly Gly Asn Ala Met Asp Leu Trp Pro Ser Met Met Phe Leu 355 360 365 Leu <110> Korea Research Institute of Bioscience and Biotechnology <120> A New Stress-Resistance Transcription Factor Gene, its Protein          and A Transfected Plant by the Gene <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 1286 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of CaPF1 full length cDNA (1286 bp) <400> 1 atgtgtggtg gtgcaattat ctccgatttg gtacctccta gccggatttc ccgccggcta 60 accgccgagt tgctatgggg taactctgat ctgagcaaaa agaagaaaaa tccagggaat 120 tattactcaa agcctttgaa caggtctaag tttattgacc ttgatgagga atttgaagct 180 gactttcagg acttcaagga ctatgccgat gacgatgttg atgatgttaa gcccttcggt 240 tccaaatctg tgaaatctgg cgattcaagc tgcgatactg aaaaatcttc caagagaaag 300 aggaagaatc agtaccgggg gatcagacag cgtccttggg gtaagtgggc agctgaaatt 360 cgtgatccga ggaaagggat tcgagtttgg cttggaactt tcaattctgc ggaagaagca 420 gctagagctt atgatgttga ggcacgaagg atcagaggca agaaggctaa ggtgaacttt 480 cctgatggat ctccagcttc tgcttcaaga cgtgctgtta agccaaatcc tcaggaggca 540 cttcgcgagg aaatcttgaa cacagttcag ccgaacacaa cttatatcaa caacttggac 600 ggcggatctg atgattcgtt tggctttttc gaagagaaac cagcagcaaa gcagtatggc 660 tatgagaatg tttcttttac tgctggagat atgggactgg gttcaatttc cccttcaact 720 ggtacaacaa atgtttactt cagttctgat gaaggaagca acacctttga ctgctctgat 780 ttcggttggg gtgaaccatg tccgaggact ccagagatct catctgttct gtcagaagtt 840 ctagaatgta atggtactca atctgatgaa gatgctagac cagagaaaaa actgaagtcg 900 tgttccaacg cttccttgcc agatgaggat aacactgtgc acacgctatc tgaagagcta 960 tcggcttttg aatcccagat gaagttcttg cagatcccat atcttgaggg aaattgggat 1020 gcatcagttg atgcctttgt caacacaggc gcaattcagg atggcggaaa tgcgatggat 1080 ctctggcctt cgatgatgtt ccttctttaa tgggaggtgt ctataagcca acacgcacct 1140 tcccttatta agttttgtaa ataaagcttc atttgagtga agtttgcagt tatgttgtct 1200 ccaaacaaaa aagactatat atgtgttgta ttaaatttat ttcataaatt tacttgtttg 1260 atgtaaaaaa aaaaaaaaaa aaaaaa 1286 <210> 2 <211> 369 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of CaPF1 <400> 2 Met Cys Gly Gly Ala Ile Ile Ser Asp Leu Val Pro Pro Ser Arg Ile   1 5 10 15 Ser Arg Arg Leu Thr Ala Glu Leu Leu Trp Gly Asn Ser Asp Leu Ser              20 25 30 Lys Lys Lys Lys Asn Pro Gly Asn Tyr Tyr Ser Lys Pro Leu Asn Arg          35 40 45 Ser Lys Phe Ile Asp Leu Asp Glu Glu Phe Glu Ala Asp Phe Gln Asp      50 55 60 Phe Lys Asp Tyr Ala Asp Asp Asp Val Asp Asp Val Lys Pro Phe Gly  65 70 75 80 Ser Lys Ser Val Lys Ser Gly Asp Ser Ser Cys Asp Thr Glu Lys Ser                  85 90 95 Ser Lys Arg Lys Arg Lys Asn Gln Tyr Arg Gly Ile Arg Gln Arg Pro             100 105 110 Trp Gly Lys Trp Ala Ala Glu Ile Arg Asp Pro Arg Lys Gly Ile Arg         115 120 125 Val Trp Leu Gly Thr Phe Asn Ser Ala Glu Glu Ala Ala Arg Ala Tyr     130 135 140 Asp Val Glu Ala Arg Arg Ile Arg Gly Lys Lys Ala Lys Val Asn Phe 145 150 155 160 Pro Asp Gly Ser Pro Ala Ser Ala Ser Arg Arg Ala Val Lys Pro Asn                 165 170 175 Pro Gln Glu Ala Leu Arg Glu Glu Ile Leu Asn Thr Val Gln Pro Asn             180 185 190 Thr Thr Tyr Ile Asn Asn Leu Asp Gly Gly Ser Asp Asp Ser Phe Gly         195 200 205 Phe Phe Glu Glu Lys Pro Ala Ala Lys Gln Tyr Gly Tyr Glu Asn Val     210 215 220 Ser Phe Thr Ala Gly Asp Met Gly Leu Gly Ser Ile Ser Pro Ser Thr 225 230 235 240 Gly Thr Thr Asn Val Tyr Phe Ser Ser Asp Glu Gly Ser Asn Thr Phe                 245 250 255 Asp Cys Ser Asp Phe Gly Trp Gly Glu Pro Cys Pro Arg Thr Pro Glu             260 265 270 Ile Ser Ser Val Leu Ser Glu Val Leu Glu Cys Asn Gly Thr Gln Ser         275 280 285 Asp Glu Asp Ala Arg Pro Glu Lys Lys Leu Lys Ser Cys Ser Asn Ala     290 295 300 Ser Leu Pro Asp Glu Asp Asn Thr Val His Thr Leu Ser Glu Glu Leu 305 310 315 320 Ser Ala Phe Glu Ser Gln Met Lys Phe Leu Gln Ile Pro Tyr Leu Glu                 325 330 335 Gly Asn Trp Asp Ala Ser Val Asp Ala Phe Val Asn Thr Gly Ala Ile             340 345 350 Gln Asp Gly Gly Asn Ala Met Asp Leu Trp Pro Ser Met Met Phe Leu         355 360 365 Leu

Claims (16)

서열번호 2에 기재된 아미노산 서열을 코딩하는, 식물 스트레스 저항성 유도 전사인자 CaPF1 유전자.A plant stress resistance inducing transcription factor CaPF1 gene encoding the amino acid sequence set forth in SEQ ID NO: 2. 삭제delete 삭제delete 삭제delete 제 1 항에 있어서,The method of claim 1, 상기 유전자는 서열번호 1의 염기서열로 구성된 것을 특징으로 하는 CaPF1 유전자.The gene is CaPF1 gene, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1. 제 1 항 또는 제 5 항 중 어느 한 항에 있어서,The method according to any one of claims 1 to 5, 상기 스트레스는 바이러스성, 세균성, 진균성 병원균이거나, 비생물적 스트레스인 것을 특징으로 하는 CaPF1 유전자.The stress is a viral, bacterial, fungal pathogen or CaPF1 gene, characterized in that the abiotic stress. 서열번호 2의 아미노산 서열을 가지는 것을 특징으로 하는 식물체 스트레스 저항성 유도 전사인자 CaPF1 단백질.Plant stress resistance induced transcription factor CaPF1 protein, characterized by having the amino acid sequence of SEQ ID NO: 2. 삭제delete 삭제delete 제 1 항 또는 제 5 항에 의한 CaPF1 유전자를 내재하고 있는 식물체 스트레스 저항성 부여를 위한 형질전환용 발현벡터.The expression vector for transformation for imparting plant stress resistance inherent in the CaPF1 gene according to claim 1 or 5. 제 10 항에 의한 형질전환용 발현벡터가 도입된 식물체 세포.Plant cells into which the expression vector for transformation according to claim 10 is introduced. 삭제delete 삭제delete 삭제delete 제 1 항 또는 제 5 항에 의한 CaPF1 유전자를 식물체에 도입하여 식물체에 스트레스 저항성을 부여하는 방법.A method for imparting stress resistance to a plant by introducing the CaPF1 gene according to claim 1 or 5 into a plant. 제 7 항에 의한 식물체 스트레스 저항성 유도 전사인자 CaPF1 단백질의 발현을 변화시켜 식물체의 스트레스 저항성을 조절하는 방법.Plant stress resistance induction transcription factor according to claim 7 to change the expression of CaPF1 protein to control the stress resistance of the plant.
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